Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor

The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09  Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R.

In panels a, b, c and f, water molecules are shown as red spheres.In panels b, c, f, g, h and i, progesterone (panels b and c) or DHEAS (panels f, g, h and i) are shown as cyan sticks, and the purple mesh shows the simulated annealing Fo-Fc omit map contoured at 3.0 s level.Residues surrounding the side-entrance are displayed in surface mode.Among these residues, R116 (b4/b5 loop), S177 (a4) and F181 (a4) are colored in blue, red and yellow, respectively.In

Supplementary Table 1. Published hs1R/ xls1R structures bound to synthetic ligands.
*The basic nitrogen atom that interacts with E169 (xls1R) or E172 (hs1R) through direct polar interactions is indicated by a green dashed circle.

Figure 2 .
Structural features of xls1Runknown-lig and the xlσ1R-progesterone complex.(a) Crystal packing of xls1Runknown-lig in the I432 space group.Each asymmetric unit contains one xls1R protomer (green cartoon).The a1 helices of a xls1R trimer are shown as red cylinders.(b) Superposition of the xls1Rprog-co trimer (yellow cartoon) and the xls1Runknown-lig trimer (green cartoon).Viewed perpendicular to the membrane from the b-barrel side.(c) Superposition of xlσ1Runknown-lig (green cartoon) onto a previously reported xlσ1R structure (xlσ1Rclosed-endo, PDB entry 7W2B, blue cartoon).The relative position of the ER membrane is indicated by a grey rectangle.(d) Superposition of a xlσ1Rprog-soak protomer (orange tube) onto xlσ1Rprog-co (yellow tube).(e) Superposition of lumen-lining residues between xls1Runknown-lig (green sticks) and xls1Rprog-co (yellow sticks).(f) Superposition of xlσ1Rprog-co (in yellow) onto the hσ1R-haloperidol structure (in lime green, PDB entry 6DJZ).Progesterone, haloperidol and residue E172/E169 are shown as sticks.The b-barrel lumen is indicated by orange surface.Supplementary Figure 3. Sequence alignment of s1R homologs by ClustalW 1,2 .Alpha helices are indicated by cylinders and labeled from a1 to a5.The b-barrel is indicated by ten arrows labeled from b1 to b10.Residues participating in polar or hydrophobic interactions with ligands are highlighted in cyan or yellow, respectively.Residue Y117 is highlighted in both cyan and yellow to reflect that it may participate in both polar and hydrophobic interactions.The highly conserved residue E169 (xls1R)/E172 (hs1R) is underscored.Asterisks (*) indicate identical residues.Colons (:) indicate strong similarities.Periods (.) indicate weak similarities.xls1R, s1R from Xenopus laevis; ms1R, s1R from Mus musculus; hs1R, s1R from Homo sapiens.Supplementary Figure 4. Water near the hinge-entrance of xlσ1R.(a) Water molecules (yellow/green spheres) near the hinge-entrance of xlσ1Rprog-co (yellow cartoon) and xlσ1Runknownlig (green cartoon).(b) The shortest distance between the hinge-entrance water (red spheres) and the distal b-barrel lumen water (red spheres) in xlσ1Rprog-co is indicated by a black dashed line.Residues along the line are shown as sticks.In both panels, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views.Supplementary Figure 5. MD simulation of xlσ1Rprog-co.(a) Snapshot of a xlσ1Rprog-co monomer showing a closed side-entrance before MD simulation.(b)-(d) Snapshots of xlσ1Rprog-co showing an open side-entrance at 54 th ns of MD run #1 (panel b), 17 th ns of MD run #2 (panel c) and 98 th ns of MD run #3 (panel d).The location of the side-entrance is indicated by a black arrow.
all panels, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views.Supplementary Figure 6.The side-open conformation of xlσ1R structures.(a) A trimer of the xlσ1Rside-open structure.Three protomers (A, B and C) are shown in different colors.Residues Y117 and W118 of one protomer and residues Q191 and F193 of the adjacent protomer are rendered in spheres.For a clearer view, a1 is not shown in this panel.(b) A close-up view of panel a, showing the contacting region between protomer A (side-closed, in green) and protomer C (side-open, in light blue).(c) A close-up view of panel a, showing the contacting region between protomer A (side-closed, in green) and protomer B (side-closed, in pink).(d) Structure of xls1Rside-open-all with each asymmetric unit containing two trimers (six protomers) in yellow orange.(e) Protomer A of xls1Rside-open-all.(f) Superposition of protomers A to F (all in yellow orange) of xls1Rside-open-all.In panels e and f, residues 114-118 (b4/b5 loop) and 177-181 (a4) are displayed in surface mode, and the location of the side-entrance is indicated by a black arrow.In panels e and f, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views.Supplementary Figure 9. Structure of xls1RDHEAS-C2.(a) Six protomers in each asymmetric unit of xls1RDHEAS-C2 with three different DHEAS binding poses.DHEAS Pose-1 in protomers A and D (light orange cartoon); DHEAS Pose-2 in protomers B, E and F (cyan cartoon); DHEAS Pose-3 in protomer C (green cartoon).(b) Superposition of xls1RDHEAS-C2 Pose-2 (in cyan) onto xls1RDHEAS-I432 (in pink).DHEAS is shown as sticks.(c)-(e) Density fitting of DHEAS Pose-1 (light orange sticks, panel c), Pose-2 (cyan sticks, panel d), and Pose-3 (green sticks, panel e) in the b-barrel lumen (orange surface) of xlσ1RDHEAS-C2 protomer A (panel c), protomer B (panel d), and protomer C (panel e).In panels c, d and e, the blue mesh shows the simulated annealing 2Fo-Fc map contoured at 1.2 s level.

Table 3 . Affinities (Kd) of two neurosteroid ligands to xls1R determined by MST.
MST measurements were repeated with N=3 biologically independent samples and source data of Kd values are shown.Two-tailed Student's t-test was performed between wild-type and E169A for each ligand, and the P values are shown.

Table 4 . Interaction/binding energies between xls1R and bound neurosteroids.
Water refers to the water molecules within the distal space of the b-barrel lumen of xlσ1R structures (See Methods). *